Functional tissue engineering (FTE) seeks to enhance tissue engineered (TE) repairs using in vivo loads and strains from normal tissues to condition the constructs to their mechanical environment prior to surgery. FTE is particularly relevant for highly loaded tissues such as menisci in the human knee. In this novel application, we will develop a real-time assessment technology to shorten the development cycle for creating TE products. We will screen, in real time, how mechanical stimuli affect gene expression. Using specialized murine mesenchymal stem cells in a mechanical stimulation system, we will address the following aims: Aim 1: Create a source of murine MSCs having dual, mutually exclusive colorimetric indicators for fibroblastic and chondrocytic programs. Expression genes that combine human type I collagen promoter (yellow fluorescent protein-YFP) and cartilage-specific promoter element of CD-RAP (cyan fluorescent protein-CFP) will be constructed and injected separately into mouse blastocysts. Separate transgenic lines will be mated, and MSCs will be harvested from doubly transgenic animals that induce chondrocytic differentiation or fibroblast phenotype. Aim 2" Develop test platforms that control mechanical signals delivered to 3-D cell-seeded constructs and that provide real-time feedback of biological and mechanical events. Finalize designs of advanced, multi-specimen testing systems to apply precisely calibrated compressive and/or tensile strains to cell-seeded specimens and that enable real-time recordings of colorimetric changes within them. Aim 3" Evaluate gene expression for murine MSCs in translucent gels under controlled strain states. After inducing MSCs to chondrocytic phenotype, test the hypotheses that application of: 1) compressive strains to cell-gel constructs will maintain CFP indicative of a cartilage-specific promoter element, 2) tensile strains will induce dedifferentiation and transition from a cyan fluorescent chondrocyte to a yellow fluorescent fibroblast, and 3) tensile strains to MSC-gel constructs already exposed to compressive strains and already expressing CFP will yield dedifferentiation to fibroblasts resulting in a mixed composition of tissue components and colors. This technology can dramatically reduce time/expense during the development phase of TE fibrocartilage.